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Bovine trichomoniasis is a reproductive illness that affects cattle causing pyometra, early to mid-pregnancy miscarriages, and lower birth rates. Tritrichomonas foetus is a flagellated protozoan which first discovered in France in 1888 and composts three phases during its lifecycle including trophozoite, cyst, and pseudocyst. In addition, several factors contributed to the prevalence of trichomoniasis and fall into three categories are management, cow, and bull-related factors. The fundamental causes of bovine trichomoniasis-related embryonic or fetal death have not been precisely determined. Immunity to T. foetus has been subjected to very little contemporary research, but a thorough assessment of earlier work has been conducted. Clinical symptoms in cattle ranged from moderate endometritis or vaginitis to acute inflammation of the entire reproductive system. In cows, pregnancy-related infections result in metritis, pyometra, early embryonic demise, and abortion; whereas in bulls, it is suspected that bovine trichomoniasis should have their preputial cavity sampled and then sent to a laboratory for positive organism identification. To date, sexual contact is the known method of transmission of T. foetus. There have not been any significant attempts to create a treatment plan for cows because the infection is self-limitation. The distinct epidemiological features of bovine trichomoniasis determine the best prevention and control measures. [ABSTRACT FROM AUTHOR]

Simple Summary: This study investigates the microbiome of bulls infected with Tritrichomonas foetus (T. foetus), a protozoan causing bovine tritrichomoniasis. It aims to describe the preputial, penile, and seminal microbiomes of chronically infected bulls and evaluate different sampling methods. Eleven bulls were sampled using dacron swabs, pizzle sticks, double-guarded swabs, and semen collection. The microbiomes were dominated by Fusobacteria, Firmicutes, Bacteroidota, Actinobacteria, and Campylobacterota, with semen samples showing the most microbial diversity. Despite differences in operational taxonomic units (OTUs) between methods, there was no significant difference in microbial richness and abundance. The study highlights that T. foetus infections may lead to shifts in the reproductive microbiome, indicating a potential dysbiosis compared to healthy bulls. These findings provide a basis for future research on T. foetus impacts on bovine reproductive health and possible treatment strategies. Tritrichomonas foetus (T. foetus), the causative agent of bovine trichomoniasis, is an obligate protozoan parasite of the bovine reproductive tract and can be found on the penis, prepuce, and distal urethra of the bull and from the cranial vagina to the oviduct in the infected cow. To date, the microbiome of bulls infected with T. foetus has not been described. The objectives of this study were to (1) describe the preputial and penile microbiome of bulls chronically infected by T. foetus, (2) describe the seminal microbiome of T. foetus-infected bulls, and (3) evaluate different collection devices that could be used for sampling. Eleven bulls naturally infected with T. foetus were utilized for the collection of samples. Samples were obtained during the process of a routine breeding soundness exam utilizing either a dacron swab, pizzle stick, double-guarded swab, or semen collection. The preputial and seminal microbiome of T. foetus-infected bulls was dominated by bacterial members of the phyla Fusobacteriota, Firmicutes, Bacteroidota, Actinobacteria, and Campylobacterota. Semen collection yielded the most microbial diversity; however, there was no significant difference between the four methods (p ≥ 0.05). This study characterizes both the preputial and seminal microbial communities of bulls chronically infected by T. foetus. [ABSTRACT FROM AUTHOR]

Background: Trichomonosis is a common infection in small animals, mostly manifesting in gastrointestinal symptoms such as diarrhea. Although oral trichomonads are also known, the species found colonizing the large intestine are more frequently detected protozoa. Methods: In the present study, four wildcats, 94 domestic cats, and 25 dogs, originating from 18 different locations in Hungary, were investigated for the presence of oral and large intestinal trichomonads based on the 18S rRNA gene and ITS2. Results: All oral swabs were negative by polymerase chain reaction (PCR). However, Tritrichomonas foetus was detected in a high proportion among tested domestic cats (13.8%) and dogs (16%), and Pentatrichomonas hominis only in two domestic cats. In addition, a novel Tritrichomonas genotype was identified in one cat, probably representing a new species that was shown to be phylogenetically most closely related to Tritrichomonas casperi described recently from mice. All positive dogs and half of the positive cats showed symptoms, and among cats, the most frequent breed was the Ragdoll. Conclusions: With molecular methods, this study evaluated the prevalence of oral and intestinal trichomonads in clinical samples of dogs and cats from Hungary, providing the first evidence of T. foetus in dogs of this region. In contrast to literature data, P. hominis was more prevalent in cats than in dogs. Finally, a hitherto unknown large intestinal Tritrichomonas species (closely related to T. casperi) was shown to be present in a cat, raising two possibilities. First, this novel genotype might have been a rodent-associated pseudoparasite in the relevant cat. Otherwise, the cat was actually infected, thus suggesting the role of a predator–prey link in the evolution of this trichomonad. [ABSTRACT FROM AUTHOR]

Tritrichomonas foetus is a protozoan parasite that causes a venereal disease in cattle limiting reproduction by abortions and sterility. The immune response against this parasite is poorly understood. Since the iron and calcium ions are important regulators of the microenvironment of the urogenital tract in cattle, we decided to evaluate the role of these divalent cations on the antigenicity of membrane proteins of T. foetus on macrophage activation as one of the first inflammatory responses towards this pathogen. Colorimetric methods and ELISA were used to detect the nitric oxide and oxygen peroxide production and expression of cytokines in culture supernatant from macrophage incubated with membrane proteins from T. foetus cultured in iron‐ and calcium‐rich conditions. qRT‐PCR assays were used to evaluate the transcript expression of genes involved in the inflammatory response on the macrophages. The membrane proteins used for in vitro stimulation caused the up‐regulation of the iNOS and NOX‐2 genes as well as the generation of NO and H2O2 in murine macrophages on a dependent way of the metal concentrations. Additionally, after stimulation, macrophages showed a considerable rise in pro‐inflammatory cytokines and a downregulation of anti‐inflammatory cytokines, as well as up‐regulation in the transcription of the TLR4 and MyD88 genes. These data suggest that membrane proteins of T. foetus induced by iron and calcium can activate an inflammatory specific macrophage response via TLR4/MyD88 signalling pathway. [ABSTRACT FROM AUTHOR]

Tritrichomonas foetus is a flagellated parasite that primarily infects the reproductive tissues of livestock, causing bovine trichomoniasis. The cytoplasmic membrane of T. foetus contains various compounds that contribute to adherence, colonization, and pathogenicity. Metronidazole (MTZ) is the main treatment for trichomoniasis, but the emergence of drug‐resistant strains is a concern due to improper use and dosing. T. foetus infection induces inflammation, and macrophages are key players in the immune response. However, our understanding of the host's immune response to T. foetus is limited, and the specific mechanisms underlying these responses are not well understood. This study aimed to investigate the impact of T. foetus surface proteins from trophozoites cultured under different sublethal MTZ conditions (MTZ‐treated T. foetus MPs) on macrophage activation. By analyzing cytokine levels and gene expression in murine macrophages, we demonstrated that MTZ‐treated T. foetus MPs induce a specific proinflammatory response. MTZ‐treated T. foetus MPs‐exposed macrophages exhibited a higher NO and H2O2 production and overexpression of iNOS and NOX‐2 genes in comparison to untreated T. foetus. Additionally, MTZ‐treated T. foetus MPs triggered a significant induction of the proinflammatory cytokines IL‐1β, IL‐6, TNF‐α, and IFN‐γ, as well as the overexpression of the TLR4, MyD88, and NF‐κB genes on murine macrophages. The study aimed to unravel the immunological response and potential proinflammatory pathways involved in T. foetus infection and MTZ stress. Understanding the immune responses and mechanisms through which T. foetus surface proteins activate macrophages can contribute to the development of new therapeutic strategies for controlling bovine trichomoniasis. [ABSTRACT FROM AUTHOR]

The parasite T. foetus causes trichomonosis in cattle but is generally asymptomatic in males. Thus, many bulls carrying the disease go unnoticed, making the detection of T. foetus in bulls an important aspect for its control. Due to drawbacks posed by its cultivation, PCR is a preferred option for diagnostic laboratories. Most published PCR protocols target the genomic region compring the 18S, 5.8S, and 28S rRNA genes and internal transcribed spacers 1 and 2 (rRNA-ITS region), homologous to that of other Tritrichomonas species. There is minimal information on alternative genetic targets and no comparative studies have been published. We compared a protocol based on the microsatellite TfRE (called H94) and five protocols based on the rRNA-ITS region (called M06, M15, G02, G05, and N02). We also designed and evaluated a novel PCR-based assay on the EF1-alpha-Tf1 gene (called V21). The analytical sensitivity and specificity assays for the PCR protocols were performed according to the World Organisation for Animal Health (OIE) directives and the comparative study was performed with a widely used PCR (M06) on clinical samples from 466 breeding bulls. V21 showed a high degree of agreement with our reference M06 (kappa = 0.967), as well as M15 (kappa = 0.958), G05 (kappa = 0.948), and H94 (kappa = 0.986). Protocols H94 and V21 appear to be good approaches for confirming clinical cases in preputial bull samples when genomic regions alternative to rRNA-ITS are required. By contrast, N02 gave false negatives and G02 false positives. [ABSTRACT FROM AUTHOR]

Bovine trichomoniasis, caused by the protozoal parasite Tritrichomonas foetus, is a highly contagious venereal disease characterised by early pregnancy loss, abortion and pyometra. Persistently infected bulls and cows are the primary reservoirs of infection in infected herds. This research investigated the prevalence of T. foetus infection in bulls from properties located across northern Australia and New South Wales. Preputial samples were collected from 606 bulls at slaughter and tested for T. foetus using the VetMAX‐Gold Trich Detection Kit (Thermo Fisher Scientific). The apparent prevalence of T. foetus infection varied between regions, with northern regions in the Northern Territory, Queensland and Western Australia showing a prevalence of 15.4%, 13.8% and 11.4%, respectively. There was some evidence of an association between infection and postcode (P = 0.06) and increasing bull age (P = 0.054). This study confirms that T. foetus infection is likely to be present in many beef breeding herds and contributing to lower than expected reproductive performance, particularly across northern Australia. [ABSTRACT FROM AUTHOR]

Tritrichomonas foetus is a flagellated venereal parasite that causes Trichomoniasis. Among various infections, Tritrichomonas infection is a major threat to animal husbandry contributing to heavy economic loss due to fetal deaths or abortions. Plants are a great source of a variety of secondary products that consist of different bioactive compounds with medicinal properties. Species of Caralluma are known to be sources of potential therapeutic molecules. However, no studies have been carried out on the anti-protozoan activity of Caralluma procumbens against Tritrichomonas foetus. Hence the effect of different concentrations of Caralluma extract on the growth and survival of Tritrichomonas foetus was examined. Methanolic extract of Caralluma procumbens, at the concentrations of 5 and 10 mg/ml inhibited the growth of Tritrichomonas foetus completely after 24 hours of incubation. A concentration of 2 mg/ml inhibited 80% growth of Tritrichomonas foetus after 48 hours of incubation with Caralluma extract. The results of the current study suggest that C. procumbens could be suitable for treatment and prevention of protozoan induced Trichomoniasis and prove to be an effective therapeutic strategy without any nonspecific effects. Further studies in this line will help in identification of effective structural bioactive components present in the methanol extract of C. procumbens. [ABSTRACT FROM AUTHOR]

• Genomes of bovine, feline and porcine Tritrichomonas foetus strains were sequenced. • A close phylogenetic relationship between bovine and porcine T. foetus was found. • Feline T. foetus was found to be clearly distinct from bovine and porcine T. foetus. • Various potential genetic markers of T. foetus were identified. Tritrichomonas foetus is a protozoan parasite that colonizes the reproductive tract of cattle as well as the gastrointestinal tract of cats. Bovine tritrichomonosis is a sexually transmitted disease whereas feline tritrichomonosis is thought to be transmitted by the fecal-oral route. Furthermore, T. foetus is known as an essentially apathogenic commensal located in the nasal cavity of pigs. Transmission of T. foetus between the different hosts has to be considered a realistic scenario that may have important implications for the epidemiology of infections and disease. In our study, we generated whole genome sequencing (WGS) data from bovine, feline and porcine T. foetus strains to investigate the genetic (dis)similarities among these diverse strains. As a reference, we used a previously released draft assembly from a bovine T. foetus strain K isolated from an infected bull in Brazil. In particular, we identified single nucleotide polymorphisms (SNPs) and the insertion-deletion (indel) variations within the genomes of the different strains. Interestingly, only a low degree of polymorphism (68 SNPs and indels) was found between the bovine and the porcine strains in terms of variants with a predicted impact of moderate or high and where one species is homozygous for one allele and the other homozygous for the other allele. Conversely, however, a 964 times higher number of such differences was detected by comparing the feline with either the bovine (65,569) or the porcine (65,615) strain. These data clearly indicated a close phylogenetic relationship between bovine and porcine T. foetus but a remarkable genetic distinctness of these two strains from the feline strain. The latter observation was confirmed by PCR-based sequencing of 20 in silico-selected indel markers and five in silico-selected SNP markers that uniformly demonstrated a relatively distant phylogenetic relationship of three independent feline T. foetus isolates in comparison to the bovine and porcine strains investigated. In summary, our comparative genome sequencing approach provided further insights into the genetic diversity of T. foetus in relation to the different host origins of the parasite. Furthermore, our study identified a large number of SNP- and indel-containing sequences that may be useful molecular markers for future epidemiological studies aimed at the elucidation of the transmission patterns of T. foetus within different host species. [ABSTRACT FROM AUTHOR]

Background: Several Tritrichomonas species have been found in mammalian hosts. Among these trichomonads T. foetus is often found in the urogenital tract of cattle and the gastrointestinal tract of the domestic cat, resulting in sexually transmitted bovine trichomonosis and fecal-orally transmitted feline trichomonosis, respectively. The aims of the current study were to molecularly characterize clinical isolates of T. foetus in cattle populations in Wyoming, South Dakota, and Montana of the United States of America and to phylogenetically analyze Tritrichomonas species of mammalian hosts. Results: DNA sequencing of rRNA genes showed over 99% identity of the newly described isolates to other bovine isolates. Further, T. foetus isolates of various mammalian hosts originated in different geographic regions worldwide were clustered into two well-defined clades by phylogenetic analysis of rRNA and cysteine protease 2 genes. Clade I consisted of isolates originated from cattle, pig, and human whereas clade II contained isolates of cat and dog. Conclusion: It is concluded that all mammalian Tritrichomonas spp. apparently belong to T. foetus. Analysis of more sequences is warranted to support this conclusion. [ABSTRACT FROM AUTHOR]

Tritrichomonas foetus has been identified in the ileum, cecum and colon of cats with large bowel diarrhea. The objective of this study was to identify whether T. foetus was present in cats with chronic diarrhea in Turkey. Fecal samples (n=45) were collected from cats with diarrhea presented to a number of pet clinics in Ankara province, Turkey. Fecal analysis was performed using saline wet mount preparations, cultures and PCR. T. foetus was detected in 4 out of 45 (8.8%) of the cats with chronic diarrhea by PCR analysis. The infection rate was highest (12%) in the age group =6 months (p<0.05). There was no significant difference between the frequency of infection in male and female cats (p>0.05). All cats in the study were mixed breed. The frequency of infection was higher in owned cats than in shelter cats (10% versus 6.6%), but this difference was not statistically significant (p>0.05). Giardia spp. and Toxocara leonina were found as co-infections in the cats infected with T. foetus. Two PCR positive results were sequenced and deposited in GenBank (MH400076 and MH490937). Pairwise genetic distance between our isolates and the isolates submitted to GenBank from other geographic areas displayed variation ranging from 0.0% to 1.2%. This is the first report of the presence of T. foetus in cats in Turkey. The results support the significance of T. foetus as a cause of chronic diarrhea in cats. [ABSTRACT FROM AUTHOR]

• Real-time PCR and LAMP assays for detection of Tritrichomonas foetus were developed. • These assays are complementary to previously established PCR and LAMP tests. • These assays are suitable for direct coprological diagnosis of tritrichomonosis. • LAMP allows diagnosis of tritrichomonosis under poorly equipped lab conditions. The protozoan parasite Tritrichomonas foetus may cause severe diarrhea in cats all over the world. In order to evaluate the methodology in coprological molecular diagnosis of feline tritrichomonosis, we compared previously published ("old") and newly developed ("novel") loop-mediated isothermal amplification (LAMP) (targeted to the T. foetus β-tubulin and the elf1α 1 gene, respectively) as well as an old conventional and an old and novel real-time PCR (all targeted to overlapping regions of T. foetus rDNA) assays regarding their diagnostic sensitivities and specificities. Here, the novel real-time PCR yielded the best methodical performance in that a sensitivity with a detection limit of < 0.1 trophozoites (corresponding to ca. < 0.13 trophozoites per mg feces) and a maximal specificity for diagnosis of Tritrichomonas spp. was achieved. The other test systems exhibited either an approximately 10-times lower sensitivity (< 1 trophozoite corresponding to ca. < 1.3 trophozoites per mg feces) (conventional PCR and both LAMP assays) or a lower specificity (old real-time PCR). Conversely, the diagnostic performance assessed with clinical fecal samples from cats demonstrated identical sensitivities (8 of 20 samples tested were positive) for the novel PCR and both LAMP assays. Diagnostic sensitivities were significantly higher than those found for the old real-time (5 positive samples) and conventional PCR (6 positive samples), respectively. Accordingly, our data suggested the novel PCR and both LAMP assays to be well suited molecular tools for direct (i.e. without including an in vitro cultivation step) coprological diagnosis of tritrichomonosis in cats. Interestingly, relative high (novel LAMP, 7 positive samples) to at least moderate (old LAMP, 6 positive samples and 1 sample with equivocal score) diagnostic sensitivities were also achieved by testing clinical samples upon simple visual inspection of colorimetric changes during the LAMP amplification reactions. Accordingly, both LAMP assays may serve as practical molecular tools to perform epidemiological studies on feline (and bovine as well as porcine) tritrichomonosis under simple laboratory conditions. [ABSTRACT FROM AUTHOR]

Bovine trichomonosis is a sexually transmitted disease considered as an important cause of early reproductive failure in beef cattle. To investigate the occurrence of the infection in different Spanish beef cattle-producing areas, retrospective data from the SALUVET veterinary diagnostic laboratory (Veterinary Faculty, Madrid, Spain) derived from the analysis of samples from beef bulls that were routinely tested for Tritrichomonas foetus infection, were compiled from 2011 to 2015. In addition, a number of potential risk factors were assessed. T. foetus was detected in 12.7% (385/3016; 95% CI: 11.5%–13.9%) of samples from bulls and in 20.7% (195/941; 95% CI: 18.1%–23.3%) of the herds tested. "Bull age" and "reproductive disorders in the herd" were the risk factors identified in the multivariable analysis. Bulls older than 3 years (19.7%) were more likely to be infected than young bulls (8.2%) and T. foetus was more often detected in herds with reproductive problems (27.9%) than in those without reproductive disorders (9.4%). The prevalence in bulls originating in mountain systems (13.9%, 267/1922) was significantly higher than that in "dehesa" (Mediterranean holm-oak pasture) areas (10.8%; 118/1094) (P ˂ 0.05), which might be attributable to the use of communal pastures and specific management practices in mountain systems. The results reported here indicate that T. foetus infection is substantially spread among beef cattle herds, suggesting that BT could be having a significant negative impact on the reproduction and productivity of Spanish beef herds managed under extensive conditions. • Presence of bovine trichomonosis in Spanish beef bulls. • Tritrichomonas foetus was detected in 12.7% of breeding bulls tested from 2011 to 2015. • Greater testing prevalence in bulls from mountain systems. • Greater risk in bulls older than 3 years and bulls from herds with reproductive disorders. [ABSTRACT FROM AUTHOR]

Highlights • Flow cytometry is a powerful tool for evaluation of in vitro drug susceptibility. • Analysis of the susceptibility to metronidazole in isolates of Tritrichomonas foetus. • First report of IC 50 of Mz at 48 h in T. foetus isolates. Abstract Bovine Trichomonosis, an endemic sexually transmitted disease in countries with extensive livestock and natural service, represents one of the most common causes of reproductive failure. 5-nitroimidazoles and their derivatives are used for its treatment, mainly metronidazole (Mz). The emergence of resistance mechanisms adopted by the parasites against the drug and failure of the treatments suggest the need to investigate susceptibility and obtain current values. The available information of in vitro susceptibility of these drugs comes from the use of a diversity of methodologies and criteria, especially observation of the mobility of the parasite under the optical microscope to evaluate its viability. These techniques are arduous and time consuming and lead to a subjective assessment dependent on the operator, the methodology used, and the morphology adopted by the protozoan. In this sense, flow cytometry has proven to be a fast and efficient method to evaluate viability in other protozoa. The aim of this study was to evaluate the in vitro susceptibility of six bovine isolates of Tritrichomonas foetus to Mz in aerobic (AC) and anaerobic (ANC) conditions by means of the calculation of the 50% inhibitory concentration (IC 50), by flow cytometry, and also to analyze minimum lethal concentration (MLC) by means of recovery tests post-treatment in vitro. IC 50 values ranged from 1.06 to 1.25 μM in ANC and from 1.44 to 3.03 μM in AC, these being the only ones reported at 48 h for these protozoa. With respect to MLC at 48 h, the results were from 3.67 to 7.35 μM in ANC, and from 7.35 to 14.7 μM for AC, where two isolates (Tf0 and Tf2) for AC and one (Tf2) for ANC showed higher values than those described in the literature. Flow cytometry has proven to be an effective, rapid and objective methodology and very useful in susceptibility tests. The data obtained through these tests allow us to describe the susceptibility exhibited by these protozoa, this being valuable information when establishing dosages in Mz treatments. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Giardia intestinalis and Tritrichomonas foetus in Small Animal Diarrhoea PCR diagnostic panel. • Multiplex tandem real-time PCR assay for feline faecal samples. • 95.1% sensitivity and 92.1% specificity for Giardia intestinalis Assemblage F DNA. • 41.9% sensitivity and 100.0% specificity for Tritrichomonas foetus genotype 'feline' DNA. • High proportion of late amplifiers for T. foetus compared to G. intestinalis positive samples. Abstract Giardia intestinalis and Tritrichomonas foetus are frequent enteric protozoan parasites of the gastrointestinal track of domestic cats. Because of different treatment options for the parasites, confirmation of presence of one or both pathogens is necessary. The PCR based assays are suitable for differential diagnosis. We evaluated the performance of Small Animal Diarrhoea panel, a multiplexed-tandem real-time PCR (MT-PCR) assay, that detects DNA of both G. intestinalis and T. foetus. The sensitivity and specificity were compared to reference real-time PCR assays using 105 faecal samples, 39.05% (n = 41) positive for G. intestinalis and 30.48% (n = 32) were positive for T. foetus. The faecal samples positive for T. foetus had a high proportion of late amplifiers, determined by an arbitrary threshold of C t -values > 35. On the other hand, only one G. intestinalis positive sample was considered a late amplifier. For G. intestinalis DNA, the MT-PCR assay had 95.1% sensitivity and 92.1% specificity. For T. foetus DNA, the MT-PCR assay had 41.9% sensitivity and 100.0% specificity. To evaluate the interlaboratory reproducibility of the MT-PCR assay, results were compared in two different laboratories and found to be in a very good agreement (Kappa = 0.9). Further analysis of the DNA using conventional PCR determined presence of G. intestinalis Assemblage F and T. foetus genotype 'feline'. In conclusion, the MT-PCR Small Animal Diarrhoea panel had a good and poor performance against reference assays for G. intestinalis and T. foetus, respectively. The assay is suitable for detection and differential diagnosis of G. intestinalis and moderate to high burdens of T. foetus in small animal clinical practice. [ABSTRACT FROM AUTHOR]

Background: Tritrichomonas foetus is recently found to be one of the causes of chronic diarrhea in domestic cats but there is no report of feline intestinal trichomoniasis in Iran. Case description: A 9 months female domestic short-haired cat was referred to our hospital with a history of chronic diarrhea over the past two months, and with an unsuccessful previous antibiotic therapy in another clinic. Findings/treatment and outcome: Clinical examination revealed 39.8°C rectal temperature. Complete blood count (CBC) showed moderate inflammatory leukogram. By the wet-mount faecal assessment, moving organisms were observed. Rectal scarping dry-mount cytology revealed many inflammatory cells along with huge numbers of trophozoites with typical morphologic characteristics including central axostyle, undulating membrane, flagella, and one laterally located nucleus. Polymerase chain reaction (PCR) was performed and confirmed the diagnosis of T. foetus. After treatment with metronidazole and probiotics, the cat was completely recovered. Conclusion: This study represents the first case of intestinal trichomoniasis in a cat with chronic diarrhea in Iran. [ABSTRACT FROM AUTHOR]

Light microscopy has significantly advanced in recent decades, especially concerning the increased resolution obtained in fluorescence images. Here we present the Expansion Microscopy (ExM) technique in two parasites, Trichomonas vaginalis and Tritrichomonas foetus , which significantly improved the localization of distinct proteins closely associated with cytoskeleton by immunofluorescence microscopy. The ExM techniques have been used in various cell types, tissues and other protist parasites. It requires the embedment of the samples in a swellable gel that is highly hydrophilic. As a result, cells are expanded 4.5 times in an isotropic manner, offering a spatial resolution of ∼70 nm. We used this new methodology not only to observe the structural organization of protozoa in more detail but also to increase the resolution by immunofluorescence microscopy of two major proteins such as tubulin, found in structures formed by microtubules, and costain 1, the only protein identified until now in the T. foetus 's costa, a unique rod-shaped like structure. The individualized microtubules of the axostyle were seen for the first time in fluorescence microscopy and several other details are presented after this technique. [Display omitted] • An innovative technique to improve the visualization of Trichomonas vaginalis and Tritrichomonas foetus cytoskeleton. • Expansion Microscopy (ExM) significantly improved the localization of distinct proteins. • Embedment of the samples in a highly hydrophilic swellable gel for improved light microscopy analysis. [ABSTRACT FROM AUTHOR]

Abstract The venereal diseases bovine trichomoniasis (BT) and bovine genital campylobacteriosis (BGC) cause economic losses in endemic areas like La Pampa province in Argentina, where beef cattle are usually managed extensively. The eradication of both diseases is a priority in Argentine. The aim of this case-control study was to identify shared and specific risk factors of both diseases from the data obtained from the Provincial Program for the Control and Eradication of BT and BGC in La Pampa (PCE). The study population included 5777 breeding beef herds tested for BT and BGC in 2013. The study comprised 3481 herds that were surveyed in situ by official PCE veterinarians, distributed as follows: 3159 negative herds (with no BT or BGC positive bulls), 121 BT case herds (with at least a BT positive bull), 170 BGC case herds (with at least a BGC positive bull) and 31 BT and BGC co-infected case herds (with at least a positive bull for each disease). Three multivariable analyses were performed in order to identify the risk factors using a logistic regression model. Results showed that the risk factors shared by both diseases were the number of bulls (OR for BT = 1.03, 95% CI: 1.02–1.04, p < 0.001; OR for BGC = 1.02, 95% CI: 1.01–1.03, p < 0.001; OR for BT and BGC co-infection = 1.02, 95% CI: 1.01–1.04, p = 0.003) and the presence in the previous year of each disease (OR for BT = 18.69, 95% CI: 11.82–29.57, p < 0.001; OR for BGC = 4.65, 95% CI: 3.18–6.81, p < 0.001; OR for BT and BGC co-infection = 39.18, 95% CI: 16.72–91.80, p < 0.001). Disease-specific risk factors were continuous breeding season for BT (OR = 1.59, 95% CI: 1.04–2.47, p = 0.034), and inadequate condition of the fences for BGC (OR = 1.51, 95% CI: 1.06–2.15, p = 0.022). An integrated approach to venereal disease management needs to consider the risk characteristics identified in this study. This could lead to reduce both diseases and improve the efficiency of BT and BGC control activities in La Pampa. [ABSTRACT FROM AUTHOR]

Highlights Fast and sensitive method for Tritrichomonas foetus detection. Based on loop mediated isothermal amplification of T. foetus elongation factor 1 alpha 1. 100 times more sensitive than culture, tf-5.8 r LAMP or TFR3-TFR4 PCR. Direct detection from smegma and cervical mucus. Abstract Testing for Tritrichomonas foetus and exclusion of infected animals is an effective way of improving the reproductive efficiency in a herd. Conventional PCR is inherently more specific than the culture method and quantitative PCR can significantly increase the detection limit. Loop Mediated Isothermal DNA Amplification (LAMP) is gaining interest because the method does not require expensive equipment, specificity and sensitivity can be as high as quantitative PCR. The object of this study was to develop a sensitive and friendly test for point-of-care detection of T. foetus. The LAMP test that targeted T. foetus elongation factor 1 alpha 1 sequences showed high specificity. Sensitivity was 100–1000 times higher than that reached through culture, polymerase chain reaction or with a previously developed LAMP for 5.8 ribosomal sequences. Moreover, T. foetus detection could be performed without DNA purification from infected cervical vaginal mucus (CVM) or smegma samples. The tf-ef1a1 LAMP method was tested for field detection with paper strips soaked in CVM from infected cows and the results were observed 90 min later. Direct detection of T. foetus in CVM with the tf-ef1a1 LAMP showed high sensitivity and specificity, and an overall diagnostic odds ratio of 56 (CI: 13.3–235.0). The tf-ef1a1 LAMP showed great potential for diagnosis and control of T. foetus in resource-challenged regions. [ABSTRACT FROM AUTHOR]

Trichomoniasis is a disease caused by protozoan parasites that live in the gastrointestinal tract or the genitourinary system. Often these protozoa are non-pathogenic, but there are also highly pathogenic species. Tritrichomonas foetus is a well-studied pathogen parasitizing the reproductive system of cattle. Recently, it was found that the same pathogen is able to parasitize in the intestines of cats - the subvascular or colon guts, causing colonic diarrhea. High risks of development of this invasion are noted in animals kept in nurseries, shelters for homeless animals. This article provides a brief overview of the state of the studies of trichomoniasis in cats and the characteristics of this parasite. The analysis of the results of studies of scientists on the detection of trichomoniasis in cats and methods of research for the detection of a parasite in feces of cats. Questions that are relevant for the prospects of development of this issue in veterinary medicine are highlighted. [ABSTRACT FROM AUTHOR]

The microaerophilic parasites Entamoeba histolytica , Trichomonas vaginalis , and Giardia lamblia jointly cause hundreds of millions of infections in humans every year. Other microaerophilic parasites such as Tritrichomonas foetus and Spironucleus spp. pose a relevant health problem in veterinary medicine. Unfortunately, vaccines against these pathogens are unavailable, but their microaerophilic lifestyle opens opportunities for specifically developed chemotherapeutics. In particular, their high sensitivity towards oxygen can be exploited by targeting redox enzymes. This review focusses on the redox pathways of microaerophilic parasites and on drugs, either already in use or currently in the state of development, which target these pathways. [ABSTRACT FROM AUTHOR]

The utility of therapeutic vaccination of bulls against Tritrichomonas foetus has been advocated in previous studies, but anecdotal reports suggest this practice does not clear infections and may additionally confound diagnostic testing by reducing parasite burdens below detectable limits. The objective of this study was to characterize the systemic humoral immune response to therapeutic vaccination in T. foetus -infected bulls over a period of four months using an indirect ELISA and to compare the dynamics of this response to culture and PCR results to establish the existence of a relationship (or lack thereof) between immunization and infection status. A study population of 4- to 6-year-old T. foetus- infected beef bulls ( n  = 20) was divided equally into a treatment group and a control group. The treatment group received two doses of commercially prepared whole cell killed vaccine 2 weeks apart while the control group received injections of vaccine diluent. Blood samples were collected at each injection and at 4 subsequent dates every 4 weeks thereafter (i.e. 0, 2, 6, 10, 14, and 18 wks) to measure IgG 1 and IgG 2 antibody subisotype response via an indirect ELISA. Preputial smegma samples were collected at the four monthly intervals following vaccination for diagnosis of infection via InPouch™ culture, Modified Diamond’s Medium (MDM) culture, and PCR. Humoral response for both IgG isotypes from week 2 through week 18 were significantly increased in vaccinates compared to controls. No significant decrease in infection prevalence was detected in the treatment group for any of the diagnostic methods used. The apparent lack of pathogen clearance during a stimulated immune response suggests that therapeutic vaccination may not be a useful T. foetus management practice. [ABSTRACT FROM AUTHOR]

Vaccines against Tritrichomonas foetus have been shown to reduce the time of infection after natural or experimental exposure. The object of this study was to assess the protection against T. foetus infection conferred by a single vaginal instillation of formaldehyde fixed T. foetus cells. Aberdeen Angus virgin heifers were randomly allocated to 3 groups of 12 individuals to receive placebo or formaldehyde fixed T . foetus cells prepared following one of two procedures (formalin or freshly prepared solution) and six weeks later they were challenged with 10 6 T. foetus trophozoites. The median time for clearance among control heifers was 93.75 days while in animals immunized with formaldehyde fixed T. foetus it was 45 days. A single vaginal dose of cells fixed with fresh formaldehyde solution gave a rate of decay of infection per unit of time of 2.54 (CI 95% = 1.07;6.01). [ABSTRACT FROM AUTHOR]

Abstract: The flagellated protist Tritrichomonas foetus is a parasite that causes bovine trichomonosis, a major sexually transmitted disease in cattle. Cell division has been described as a key player in controlling cell survival in other cells, including parasites but there is no information on the regulation of this process in T. foetus. The regulation of cytokinetic abscission, the final stage of cell division, is mediated by members of the ESCRT (endosomal sorting complex required for transport) machinery. VPS32 is a subunit within the ESCRTIII complex and here, we report that TfVPS32 is localized on cytoplasmic vesicles and a redistribution of the protein to the midbody is observed during the cellular division. In concordance with its localization, deletion of TfVPS32 C‐terminal alpha helices (α5 helix and/or α4‐5 helix) leads to abnormal T. foetus growth, an increase in the percentage of multinucleated parasites and cell cycle arrest at G2/M phase. Together, these results indicate a role of this protein in controlling normal cell division. [ABSTRACT FROM AUTHOR]

Trichomonas vaginalis and Tritrichomonas foetus are pathogens that parasitise, respectively, human and bovine urogenital tracts causing disease. Using LC-MS, reference metabolomic profiles were obtained for both species and stable isotope labelling with D-[U-13C6] glucose was used to analyse central carbon metabolism. This facilitated a comparison of the metabolic pathways of T. vaginalis and T. foetus, extending earlier targeted biochemical studies. 43 metabolites, whose identities were confirmed by comparison of their retention times with authentic standards, occurred at more than 3-fold difference in peak intensity between T. vaginalis and T. foetus. 18 metabolites that were removed from or released into the medium during growth also showed more than 3-fold difference between the species. Major differences were observed in cysteine and methionine metabolism in which homocysteine, produced as a bi-product of trans-methylation, is catabolised by methionine γ-lyase in T. vaginalis but converted to cystathionine in T. foetus. Both species synthesise methylthioadenosine by an unusual mechanism, but it is not used as a substrate for methionine recycling. T. vaginalis also produces and exports high levels of S-methylcysteine, whereas only negligible levels were found in T. foetus which maintains significantly higher intracellular levels of cysteine. 13C-labeling confirmed that both cysteine and S-methylcysteine are synthesised by T. vaginalis; S-methylcysteine can be generated by recombinant T. vaginalis cysteine synthase using phosphoserine and methanethiol. T. foetus contained higher levels of ornithine and citrulline than T. vaginalis and exported increased levels of putrescine, suggesting greater flux through the arginine dihydrolase pathway. T. vaginalis produced and exported hydroxy acid derivatives of certain amino acids, particularly 2-hydroxyisocaproic acid derived from leucine, whereas negligible levels of these metabolites occurred in T. foetus. [ABSTRACT FROM AUTHOR]

Background: Tritrichomonads like porcine Tritrichomonas foetus (previously named Tritrichomonas suis), can commensally live in nasal cavity of pigs, but it is rare to cause pulmonary tritrichomonosis. Case presentation: A 40-day-old piglet was presented for persistent labor breathing and diagnosed with parasite infections in the lung by analysis of bronchoalveolar lavage (BAL) under microscope. By taking advantage of next-generation sequencing approach, we found 9611 homologous tags belonging to 50 annotated genes of tritrichomonads by analysis of mRNA of the bronchoalveolar lavage with the parasite infection. Furthermore, RT-PCR and DNA sequencing analysis confirmed the presence of the tritrichomonad. Findings: Here, we report a case of pulmonary tritrichomonosis in a pig. By taking advantage of next-generation sequencing approach, we found 9611 homologous tags belonging to 50 annotated genes of tritrichomonads by analysis of mRNA of the bronchoalveolar lavage with the parasite infections. Furthermore, RT-PCR and DNA sequencing analysis confirmed the presence of the tritrichomonad. Conclusion: Our results demonstrate that tritrichomonads like porcine Tritrichomonas foetus can cause lung infections of pigs and reveal that next-generation sequencing is potential to identify rare diseases like pulmonary tritrichomonosis in clinical. [ABSTRACT FROM AUTHOR]

Human and bovine trichomoniasis are sexually transmitted diseases (STD) caused by Trichomonas vaginalis and Tritrichomonas foetus, respectively. Human trichomoniasis is the most common non-viral STD in the world and bovine trichomoniasis causes significant economic losses to breeders. Considering the significant impact of the infections caused by these protozoa and the treatment failures, the search for new therapeutic alternatives becomes crucial. In this study the effect of diamines and amino alcohols in the in vitro viability of trichomonads was evaluated. Screening demonstrated the high activity of diamine 4 against these protozoa. Although cytotoxicity against HMVII cell line and slight hemolysis were observed in vitro , the compound showed no toxic effect on the Galleria mellonella in vivo model. Importantly, diamine 4 was active against both trichomonads species at 6 h and 24 h of incubation, and these effects was reverted by putrescine, a polyamine, suggesting competition for the same metabolic pathway. These findings indicate that the mechanism of action of diamine 4 is through the polyamine metabolism, a pathway distinct from that presented by metronidazole, the drug usually used to treat trichomoniasis and to which resistance is widely reported. These data demonstrate the importance of diamines as potential novel candidates as anti- T. vaginalis and anti- T. foetus agents. [ABSTRACT FROM AUTHOR]

Trichomonosis has been assessed according to the criteria of the Animal Health Law (AHL), in particular criteria of Article 7 on disease profile and impacts, Article 5 on the eligibility of trichomonosis to be listed, Article 9 for the categorisation of trichomonosis according to disease prevention and control rules as in Annex IV and Article 8 on the list of animal species related to trichomonosis. The assessment has been performed following a methodology composed of information collection and compilation, expert judgement on each criterion at individual and, if no consensus was reached before, also at collective level. The output is composed of the categorical answer, and for the questions where no consensus was reached, the different supporting views are reported. Details on the methodology used for this assessment are explained in a separate opinion. According to the assessment performed, trichomonosis can be considered eligible to be listed for Union intervention as laid down in Article 5(3) of the AHL. The disease would comply with the criteria as in sections 3, 4 and 5 of Annex IV of the AHL, for the application of the disease prevention and control rules referred to in points (c), (d) and (e) of Article 9(1). The animal species to be listed for trichomonosis according to Article 8(3) criteria is cattle as susceptible and reservoir. [ABSTRACT FROM AUTHOR]

Tritrichomonas foetus ( T. foetus ) is a flagellated protozoan parasite that is recognized as a significant cause of diarrhea in domestic cats with a prevalence rate as high as 30%. No drugs have been shown to consistently eliminate T. foetus infection in all cats. Cysteine proteases (CPs) have been identified as mediators of T. foetus -induced adhesion-dependent cytotoxicity to the intestinal epithelium. These CPs represent novel targets for the treatment of feline trichomonosis. However, cats also produce CPs that are part of life-critical systems. Thus, parasitic CPs need to be selectively targeted to reduce the potential for host toxicity. Previous studies have demonstrated the importance of a specific CP, CP30, in mediating bovine and human trichomonad cytopathogenicity. This CP has also recently been identified in feline T. foetus , although the function of this protease in the feline genotype remains unknown. Therefore, the study objectives were to characterize the presence of CP30 in feline T. foetus isolates and to evaluate the effect of targeted inhibition of CP30 on feline T. foetus -induced adhesion dependent cytotoxicity. The presence of CP30 in feline T. foetus isolates was identified by In gel zymography and proteomic analysis, indirect immunofluorescence (IF), and flow cytometry using a rabbit polyclonal antibody that targets bovine T. foetus CP30 (α-CP30). The effect of inhibition of CP30 activity on T. foetus adhesion and cytotoxicity was determined using CFSE-labeled feline T. foetus and crystal violet spectrophotometric assays in a previously validated co-culture model. CP30 expression was confirmed in all feline T. foetus isolates tested by all assays. Targeted inhibition of feline T. foetus CP30 resulted in decreased T. foetus adhesion to and cytotoxicity towards IPEC-J2 monolayers compared to rabbit IgG-treated T. foetus isolates. These studies establish that CP30 is expressed by feline T. foetus isolates and may be an important virulence factor in the cytopathogenicity of feline T. foetus . The results of these studies provide strong evidence-based justification for investigation of CP30 as a novel target for the treatment of feline trichomonosis. [ABSTRACT FROM AUTHOR]

Tritrichomonas foetus ( T. foetus ) is a flagellated protozoa that infects the distal ileum and proximal colon of domestic cats, as well as the urogenital tract of cattle. Feline trichomonosis is recognized as a prevalent cause of chronic diarrhea in cats worldwide. The suspected route of transmission is fecal-oral, with cats in densely crowded environments at highest risk for infection. Thus, the recommended strategy for minimizing spread of infection is to identify and isolate T. foetus -positive cats from the general population. Rapid identification of infected cats can be challenging due to the inability to accurately and quickly detect the organism in samples at point of care facilities. Thus, identification of targets for use in development of a novel diagnostic test, as well as a vaccine or therapy for T. foetus infection is a significant area of research. Despite a difference in organ tropism between T. foetus genotypes, evidence exists for conserved virulence factors between feline and bovine T. foetus . The bovine T. foetus surface antigen, TF1.17, is an adhesin that is conserved across isolates. Vaccination with the purified antigen results in amelioration of cytopathogenicity and more rapid clearance of infection in cattle. We previously showed that three feline isolates of T. foetus were positive for TF1.17 antigen so we further hypothesized that TF1.17 is conserved across feline T. foetus isolates and that this antigen would represent an attractive target for development of a novel diagnostic test or therapy for feline trichomonosis. In these studies, we used monoclonal antibodies previously generated against 1.15 and 1.17 epitopes of the bovine T. foetus TF1.17 antigen, to evaluate for the presence and role of TF1.17 in the cytopathogenicity of feline T. foetus . A previously validated in vitro co-culture approach was used to model feline T. foetus infection. Immunoblotting, immunofluorescence assays, and flow cytometric analysis confirmed the presence and surface localization of antigen TF1.17 across all feline T. foetus isolates tested. Antigen TF1.17 was notably absent in the presumably nonpathogenic intestinal trichomonad, Pentatrichomonas hominis , a parasite that can be confused microscopically with T. foetus . Similar to bovine trichomoniasis, TF1.17 was found to promote T. foetus adhesion to the intestinal epithelium. These results support further characterization and development of the TF1.17 antigen as a possible target for the diagnosis and prevention of feline T. foetus infection. [ABSTRACT FROM AUTHOR]

Tritrichomonas foetus is a flagellated protozoan parasite that causes inflammation of the reproductive tract leading to early embryonic death and abortion in cattle, thereby resulting in significant economic losses. Testing and culling infected bulls is an important strategy for parasite control. Routine testing is mainly limited to bulls that are traveling across state lines or within states that have specific control programs. Both culture and PCR detection methods are available, but they are not typically conducted as part of a yearly breeding soundness program and are not easily conducted in the field. In the present study, we developed a bead agglutination assay for detection of T. foetus antigens. Our experiments revealed that latex beads conjugated to T. foetus lipophosphoglycan-binding antibodies visibly clump in the presence of T. foetus . The detection limit of the assay, determined using both field and laboratory isolates of the parasite, was 0.25 μg/mL and 1.0 μg/mL total T. foetus antigen, respectively. Our results indicate that an antigen detection test could offer a tool for screening bulls under field conditions. [ABSTRACT FROM AUTHOR]

The prevalence of bovine trichomonosis (BT) in TN bulls was estimated through both active screening of bulls and review of previous laboratory records. During the active bull screening, preputial smegma specimens were collected from 458 TN beef bulls at 2 cattle slaughterhouses and 2 stockyards, which serve most beef bulls in TN, between March 2014 and June 2015. Each specimen was cultured for Tritrichomonas foetus ( T. foetus ) as well as evaluated microscopically every other day for seven days for any protozoa resembling T. foetus. An aliquot of the culture media from each specimen was used for DNA extraction and subsequent qPCR testing. Two specimens were considered suspect on microscopic evaluation, but all specimens were negative for T. foetus on qPCR. This suggests that the 2 specimens were most likely contaminated by fecal trichomonads. Retrospectively, 1979 T. foetus test records from 2 major TN diagnostic laboratories were reviewed between October 2013 and September 2016. True prevalence of BT in TN beef bulls was estimated at < 0.01% from the laboratory records, although the county prevalence differed in 2 TN counties (Marshal: 0.09% and Bedford: 0.5%). Overall, the prevalence of BT in TN is low, and the current screening efforts to help control BT disease in TN are acceptable. Future efforts should focus on educating cattle stakeholders on the importance of optimal specimen collection and handling as well as routine testing for BT before cattle movement. In addition, cattle producers should be reminded of leading risk factors associated with BT in cattle. [ABSTRACT FROM AUTHOR]

Background Information The costa is a prominent striated fibre that is found in protozoa of the Trichomonadidae family that present an undulating membrane. It is composed primarily of proteins that have not yet been explored. In this study, we used cell fractionation to obtain a highly enriched costa fraction whose structure and composition was further analysed by electron microscopy and mass spectrometry. Results Electron microscopy of negatively stained samples revealed that the costa, which is a periodic structure with alternating electron-dense and electron-lucent bands, displays three distinct regions, named the head, neck and body. Fourier transform analysis showed that the electron-lucent bands present sub-bands with a regular pattern. An analysis of the costa fraction via one- and two-dimensional electrophoresis and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) allowed the identification of 54 hypothetical proteins. Fourteen of those proteins were considered to be major components of the fraction. Conclusions The costa of T. foetus is a complex and organised cytoskeleton structure made of a large number of proteins which is assembled into filamentous structures. Some of these proteins exhibit uncharacterised domains and no function related according to gene ontology, suggesting that the costa structure may be formed by a new class of proteins that differ from those previously described in other organisms. Seven of these proteins contain prefoldin domains displaying coiled-coil regions. This propriety is shared with proteins of the striated fibres of other protozoan as well as in intermediate filaments. Significance Our observations suggest the presence of a new class of the cytoskeleton filaments in T. foetus. We believe that our data could auxiliate in determining the specific locations of these proteins in the distinct regions that compose the costa, as well as to define the functional roles of each component. Therefore, our study will help in the better understanding of the organisation and function of this structure in unicellular organisms. [ABSTRACT FROM AUTHOR]

Tritrichomonas blagburni (previously called T. foetus) has been implicated as an aetiological agent of long-term large-bowel diarrhoea in cats in many countries worldwide. The aim of this study was to determine the presence of, and risk factors for T. blagburni among a cohort of cats living in different conditions in the Czech Republic. Samples were collected from 170 cats living in different environments. The InPouchTM TF-Feline medium method was used for diagnosis of feline trichomonosis. A single case (0.6%) with motile trichomonads identified as Pentatrichomonas hominis was found in a cat from a multi-cat household. Our study suggests that trichomonads and in particular, T. blagburni, infection may be much less common in the Czech Republic than in neighbouring countries, despite the inclusion of cats that were likely to be from higher-risk groups. A review of studies of the association of trichomonads and feline diarrhoea carried out in different countries revealed variation in the frequency of trichomonads detected. Different combinations of PCR or culture methods for screening or confirmation have been utilised, with or without species differentiation; however, this could not solely account for the variation in the occurrence between countries. From those studies where differentiation was performed, we calculated from the combined studies that T. blagburni occurred in six cats without diarrhoea (1.1%) and 47 cases with diarrhoea (5%). This finding supports an association with diarrhoea as well as the occurrence of asymptomatic cases. We note that in many studies, including our own, the occurrence of T. blagburni may well be underestimated and suggest that future studies use a combination of PCR screening of both faeces and faecal cultures, with differentiation of trichomonad species. [ABSTRACT FROM AUTHOR]

Cows on an alpine pasture were presented with severe signs of vaginitis. To rule out infection with Tritrichomonas foetus , vaginal swabs were taken and real-time PCR based on detection via fluorescence resonance energy transfer (FRET) probes and targeting the first internal transcribed spacer (ITS-1) of nuclear ribosomal DNA (rDNA) was performed. PCR was positive in 25 of totally 34 assessed cows. However, the melting profiles of the probes targeting the diagnostic PCR products differed from the T. foetus positive control. Subsequent sequencing of the amplicons revealed 91% identity to Simplicimonas sp. sequences deposited in GenBank™. Furthermore, there was no clear association between positive PCR result and presence of vaginitis. To investigate the distribution of this Simplicimonas -like organism in cows, more herds grazing on the same alpine pastures as well as unrelated cows were tested. In total, 133 cows and 16 heifers were sampled, 53 cows and 6 heifers even twice. Vaginitis was evident in 43 cows and 4 heifers. All-over-positivity of PCR was 44%, including nine tests performed on heifers. Melting peak analysis indicated Simplicimonas -like organisms in all positive samples. Culture attempts in bovine InPouch ™ TF failed. No association between a positive PCR result and the presence of vaginitis was found. This is, to the best of our knowledge, the first report on Simplicimonas -like DNA in vaginal swabs of female cattle. Our data suggest that when testing vaginal swabs of cattle by means of T. foetus PCR, false positive reactions due to Simplicimonas -like organisms may occur. [ABSTRACT FROM AUTHOR]

Certain clinical isolates of Tritrichomonas foetus infect the urogenital tract of cattle while others infect the gastrointestinal tract of cats. Previous studies have identified subtle genetic differences between these isolates with the term “genotype” adopted to reflect host origin. The aim of this work was to seek evidence of host-specific adaptation and to clarify the relationship between T. foetus genotypes. To do this we characterised the proteomes of both genotypes using two-dimensional gel electrophoresis (2DE) coupled with LC-MS/MS. Our comparative analysis of the data revealed that both genotypes exhibited largely similar proteoform profiles; however differentiation was possible with 24 spots identified as having a four-fold or greater change. Deeper analysis using 2DE zymography and protease-specific fluorogenic substrates revealed marked differences in cysteine protease (CP) expression profiles between the two genotypes. These variances in CP activities could also account for the pathogenic and histopathological differences previously observed between T. foetus genotypes in cross-infection studies. Our findings highlight the importance of CPs as major determinants of parasite virulence and provide a foundation for future host-parasite interaction studies, with direct implications for the development of vaccines or drugs targeting T. foetus . [ABSTRACT FROM AUTHOR]

Tritrichomonas foetus is a sexually transmitted reproductive pathogen of cattle that causes transient infertility, early embryonic death, metritis, pyometra, and sporadic abortions. The objective of this research was to assess the impact on reproductive health of vaccinating naïve heifers with a killed T. foetus vaccine (TrichGuard) before experimental exposure followed by breeding. A total of 40 beef heifers were randomly assigned into two treatment groups. Heifers where then vaccinated with two doses of TrichGuard or sham vaccinated with 0.9% sterile saline according to their respective groups. Sixty days following vaccination or sham vaccination, heifers were intravaginally inoculated with 2 × 10 6 organisms of a cloned isolate of T. foetus of bovine origin (CDTf-4) during synchronized estrus. Three days following inoculation of T. foetus , bulls free of T. foetus were introduced for natural breeding. Three bulls were maintained with the 40 heifers (20 vaccinated; 20 sham vaccinated) for a 49-day breeding season. Cervical mucous samples were obtained from each heifer at Day 0 and at 29 additional time points throughout the study for T. foetus culture. Pregnancy assessments were performed routinely by using transrectal palpation and ultrasonography. Pregnancies were detected in 19/20 (95%) vaccinated heifers and 14/20 (70%) sham-vaccinated heifers (P = 0.046). Only 4/20 (20%) of the sham-vaccinated heifers gave birth to a live calf compared with 10/20 (50%) of the vaccinated heifers (P = 0.048). Thus, embryonic or fetal loss was detected in 9/19 (47%) vaccinated heifers and 10/14 (71%) sham-vaccinated heifers (P = 0.153). The interval of time between inoculations with T. foetus and conceptions of pregnancies that were maintained until birth did not differ significantly between groups (vaccinated = 18.7 days; sham-vaccinated = 17.3 days; P = 0.716). The infectious challenge in this study proved to be very rigorous as a positive culture was detected from all heifers. The culture-positive results on the last culture day did not differ significantly (P = 0.115) between vaccinated heifers (63.9 days) and sham-vaccinated heifers (79.2 days). All uterine culture samples collected from the 26 nonpregnant heifers on Day 207 postinoculation did not result in the detection of T. foetus . These findings indicate that the killed, whole cell vaccine used in this study (TrichGuard) was effective in improving reproductive health evidenced by significantly reducing losses associated with T. foetus infections. [ABSTRACT FROM AUTHOR]

Tritrichomonas foetus is a flagellated protozoan that causes a sexually transmitted disease in cattle. Trichomonosis is characterized by early abortions, subfertility and a significant decrease in productivity. Vaccine preparations containing whole T. foetus can reduce the time of residence of the pathogen in the host cervix after experimental infection. Here, T. foetus vaccines prepared with different adjuvants were tested, in parallel with a commercial vaccine, for their efficacy to clear the infection. The median time for clearance of infection was 69 days in non-immunized animals, 55 days in animals treated with aluminum hydroxide, 41 days with oil-in-water or saponin based vaccines or with a commercial vaccine and 27 days in animals treated with saponin plus aluminum hydroxide. A slight increase in the risk of T. foetus clearance from the genital tract was found with the saponin based vaccine (hazard ratio, 2.52; 95% confidence interval, 1.03–6.17) or the commercial vaccine (hazard ratio, 2.61; 95% confidence interval, 1.07–6.38). A significant increase in the risk of T. foetus clearance was found with the combination of saponin plus aluminum hydroxide based vaccine (hazard ratio, 5.12; 95% confidence interval, 2.04–12.83). [ABSTRACT FROM AUTHOR]

Tritrichomonas foetus infects the bovine urogenital tract, causing bovine trichomoniasis. Significant economic losses may occur due to infertility and abortion among cattle. Trichomonas vaginalis is the causative agent of trichomoniasis; the most common but overlooked non-viral sexually transmitted disease worldwide. Human and bovine trichomoniasis present treatment restrictions and efforts to identify new alternatives are essential. The present study evaluated the anti-trichomonads activities of seven fractions from northwest endemic plant Manilkara rufula . Flavonoids and condensed tannins were identified from these fractions by LC-DAD-MS/MS and MALDI-MS/MS. Altogether, the results demonstrated for the first time the structural description of tannins from leaves of M. rufula and the relation of these compounds with anti- T. vaginalis and anti- T. foetus activities. Overall, this report reveals the potential of M. rufula fractions against both parasites and shows new alternatives to treat the infection caused by trichomonads. [ABSTRACT FROM AUTHOR]

Bovine trichomonosis (BT), a disease of the bovine urogenital tract, is caused by the protozoan Tritrichomonas foetus (Tf). Tf causes endometritis, infertility, and premature death of the embryo, which generates considerable economic losses. The proteins released can mediate fundamental interactions between the pathogen and the host, triggering factors associated with the symptomatology, immune evasion and pathogenesis characteristic of the species. However, little is known about the profile of the proteins released by Tf. In order to contribute to their knowledge, we performed an isolation protocol and a proteomic profiling of the supernatant (SN) content of six Tf isolates. A total of 662 proteins present in the SN of Tf were detected, out of which 121 were shared by the six isolates, while the remaining 541 were found in at least one of the isolates studied. The comparative analyses using the databases of Tf strain genome K revealed 32.9% of uncharacterized proteins. The bioinformatic analyses showed that the main molecular functions predicted were binding (47.9%) and catalytic activity (38.2%). Additionally, we performed immunodetection assays to evidence the antigenic potential of SN proteins. Interestingly, we observed great ability to detect SN proteins from all six isolates using serum from immunized mice and infected bulls. A complementary mass spectrometry assay allowed us to determine that the proteins that showed the strongest signal intensity in the immunoassays were Grp78 (A0A1J4IZS3) and Ap65 (A0A1J4JSR1). This work represents the first proteomic characterization of Tf SN proteins and their antigenic potential, which might be interesting for the future design of new diagnosis and treatment methods for BT. [Display omitted] • First massive characterization of proteins released in vitro by T. foetus. • Grp78 and Ap65 proteins showed the highest signal intensity in immunoassays. • The released proteins are potential targets for the design of new diagnostic methods. [ABSTRACT FROM AUTHOR]

The objective of this study was to determine the level and duration of IgG antibodies induced against killed whole Tritrichomonas foetus and T foetus –purified surface antigen (TF1.17) in serum, vaginal, and uterine secretions after systemic immunization of beef cows with a vaccine containing killed whole T foetus. Twenty nonpregnant beef cows were randomly assigned to vaccine or control groups as follows: Vaccine (n = 10): cows received 2 mL of a commercial vaccine containing killed whole T foetus subcutaneously and a 2-mL booster 2 weeks later. Control (n = 10): cows received 2 mL of sterile saline on the same schedule. Vaginal secretions and blood samples were collected on Days 0, 8, 15, 22, 29, 36, 43, 50, 60, 75, 89, 110, 146, and 182 relative to day of primary vaccination. Uterine flush fluid was collected on Days 0, 15, 29, and 43 after the day of primary vaccination. Samples were assayed for IgG antibodies to the killed whole T foetus and surface antigen TF1.17 using enzyme-linked immunosorbent assay. Serum whole T foetus –specific IgG levels were significantly increased (between Days 15 and 182) following vaccination with T foetus or with saline. No differences between vaccinates and controls in uterine responses to whole-cell antigen were detected. Serum anti-TF1.17 IgG responses to vaccination were significantly higher than Day 0 throughout the immunization period (P < 0.001) and were higher than responses in control animals on each day post immunization through Day 146 (P < 0.001). A significant rise in TF1.17-specific IgG levels was observed in vaginal and uterine fluids from Day 15 post vaccination compared to the Day 0 levels. These levels remained significantly elevated in vaginal and uterine fluids through Days 75 (P < 0.05) and 43 (P < 0.001) after primary vaccination, respectively. Antibody levels in serum, vaginal, and uterine secretions against TF1.17 remained low in the control group throughout the study. In conclusion, vaccination of beef cows with a commercial vaccine containing T foetus induced significant increase in the levels of IgG to the T foetus TF1.17 surface antigen in serum, vaginal secretions, and uterine fluid, which remained elevated through Days 43, 75, and 182 in uterine fluids, vaginal secretions, and serum, respectively. Since purified TF1.17 antigen has been shown to protect against experimental T foetus infection in heifers, the vaccine-induced TF1.17-specific IgG response is likely to be important in the prevention of trichomoniasis in beef cattle. [ABSTRACT FROM AUTHOR]

A Tritrichomonas foetus and Giardia duodenalis mixed infection was diagnosed in two Maine Coon cats aged six months. One of them presented a history of chronic liquid diarrhea and of several unsuccessful treatments. In both cats, G. duodenalis and trichomonads were detected in fecal smears from freshly voided feces; the presence of T. foetus was confirmed by a real-time PCR assay. The cats completely recovered after treatment with ronidazole. In a refrigerated fecal sample collected from the cat with chronic diarrhea, drop-shaped trichomonad pseudocysts smaller than G. duodenalis cysts were detected. They appeared brownish or light-bluish when stained with Lugol’s solution or with Giemsa stain, respectively, and their morphological features were similar to those expressed by bovine T. foetus pseudocysts in vitro. Existence of pseudocysts even in feline trichomonads is noteworthy as they could represent a form of protozoan resistance due to unfavorable conditions whose detection in refrigerated feces can be a useful clue for clinicians. [ABSTRACT FROM AUTHOR]

Proteasomal proteolysis is required for a wide range of cellular processes, including protein quality control, cell cycle progression, cell death and metabolic adaptation to environment changes or stress responses. Proteasome inhibitors are useful compounds for determining the roles of proteasome in eukaryotic cells. Here, we investigated the effects of gliotoxin, a proteasome inhibitor, on the cell growth, replication, ultrastructure, DNA integrity and proteasomal proteolytic activity of the protist parasite Tritrichomonas foetus. The effect of gliotoxin on the transformation of T. foetus to endoflagellar form (EFF), also known as pseudocyst, was investigated. Gliotoxin inhibited the culture growth, arrested cell cycle, and provoked a trichomonacidal effect in a dose-dependent manner. Parasites treated with gliotoxin displayed features typical of cell death, such as membrane blebbing, concentric membrane whorls containing remnants of organelles, intense cytosolic and nuclear vacuolisation, chromatin condensation, DNA fragmentation, cytoplasmic disintegration and plasma membrane disruption. The proteasomal peptidase activity was inhibited by gliotoxin in a dose-dependent manner. Gliotoxin treatment also induced an irreversible EFF transformation in a dose/time-dependent manner. We compared morphological characteristics between gliotoxin- and cold-induced EFF parasites. Our results suggest that gliotoxin could induce EFF transformation by a mechanism distinct from that provoked by cold temperature. This study further contributes to a better understanding of the role of proteasome system in cell cycle, cell death and EFF transformation in T. foetus. [ABSTRACT FROM AUTHOR]

Tritrichomonas foetus is a sexually transmitted protozoan parasite that causes abortions in cattle and results in severe economic losses. In the United States, there are no safe and effective treatments for this parasite and infected animals are typically culled. In order to expedite drug discovery efforts, we investigated in vitro trophozoite killing assays amenable to high-throughput screening in 96 well plate formats. We evaluated the reduction of resorufin, incorporation of propidium iodide, and a luminescence-based ATP detection assay. Of these methods, reduction of resorufin was found to be the most reliable predictor of trophozoite concentrations. We further validated this method by conducting dose-response experiments suitable for calculation of EC 50 values for two established compounds with known activity against trophozoites in vitro , namely, metronidazole and ronidazole. Our results demonstrate that the resorufin method is suitable for high-throughput screening and could be used to enhance efforts targeting new treatments for bovine trichomoniasis. [ABSTRACT FROM AUTHOR]

Tritrichomonas foetus is a protozoan parasite that has been recently identified as a causative agent of chronic diarrhea in domestic cats. Transmission of infection occurs by the fecal-oral route through direct contact among animals. Consequently, feline trichomonosis (FT) is more likely to be present in multi-cat environments. The objective of this work was to study the presence of T. foetus and some associated risk factors in cats from densely housed origins and with a reported history of chronic diarrhea. Animals enrolled in this study were family cats (n = 15) acquired from pet shops, shelters or breeding centers and cattery cats belonging to one breeding center (n = 28) and two cat shelters (A and B, n = 25 each). In the catteries, a follow-up analysis for a period of up to 2 months was also performed to determine the parasite shedding pattern in feces and the incidence of infection. Fecal samples were analyzed using in vitro culture and a PCR technique. T. foetus was detected in a total of 38.7% (36/93) of the cats with chronic diarrhea. Parasite infection was similarly detected in family cats and cattery animals (40% versus 38.4%). In the catteries, the parasite was detected in 50%, 44% and 20% of the animals from the breeding center and shelters A and B, respectively. The follow-up analysis showed that 58.3% of infected cats intermittently shed trophozoites in their feces, with an incidence of 23.1%. Investigation of potential risk factors showed that cats ≤1 year old were more likely to be infected than older cats (57.1% versus 27.3%; P < 0.05). No significant differences were found when sex and breed factors were studied. These results confirm the importance of FT as a cause of chronic diarrhea in cats and highlight the relevance of close contact conditions for T. foetus transmission. [ABSTRACT FROM AUTHOR]

Tritrichomonas foetus is a flagellated protozoan parasite that colonizes the feline colon causing colitis and chronic foul smelling diarrhoea. Despite the efficacy of Ronidazole in the treatment of T. foetus , Ronidazole has been reported to cause neurotoxicity in some cats due to rapid absorption in the small intestine. A novel amphoteric derivative of chitosan was synthesised and characterized. A combination of time, pH, and an enzyme controlled system was used in a study of a new compression coated tablet for delivery of Ronidazole to the colon. Axial, radial swelling and erosion of selected tablets were carried out in various media. The effect of weight ratio, enzyme and pH on in vitro drug release profile was investigated. The results show that less than 2% of the drug was released in the physiological environment of the stomach and small intestine. [ABSTRACT FROM AUTHOR]

The objective is to discuss sexually transmitted diseases caused by Tritrichomonas foetus ( T foetus ) and Campylobacter fetus ( C fetus ) subsp. venerealis , with a focus on prevalence, pathogenesis, and diagnosis in cows and bulls. Diagnosis and control are problematic because these diseases cause severe reproductive losses in cows, but in bulls are clinically asymptomatic, which allows the disease to flourish, especially in the absence of legislated control programs. We review research regarding prophylactic systemic immunization of bulls and cows with antigens of T foetus and C fetus venerealis and their efficacy in preventing or clearing preexisting infections in the genital tract. Current diagnostic methods of C fetus venerealis and T foetus (microbial culture and PCR) should be improved. Review of the latest advances in bovine trichomoniasis and campylobacteriosis should promote knowledge and provide an impetus to pursue further efforts to control bovine sexually transmitted diseases. [ABSTRACT FROM AUTHOR]

Almost 20 years has passed since trichomonosis was first recognized as a potential cause of diarrhea in domestic cats. Despite progress in confirming disease causation, developing means for diagnosis, and identifying approaches to treatment of the infection, we still know very little about how this parasite causes diarrhea. With increasing recognition of resistance of trichomonosis to treatment with 5-nitroimidazole drugs, new treatment strategies based on an understanding of disease pathogenesis are needed. In this review, lessons learned from the pathogenesis of venereal trichomonosis in people and cattle are applied to clinical observations of trichomonosis in cats in effort to generate insight into areas where further research may be beneficial. [ABSTRACT FROM AUTHOR]

Objective Investigate the presence of infectious reproductive disease pathogens in dairy herd bulls in south-west Victoria, Australia, using a cross-sectional study. Methods Dairy herd bulls from 32 herds were sampled for bovine viral diarrhoea virus (BVDV: 256 bulls, 32 herds) prior to the natural mating period, bovine herpes virus-1 prior to (10 bulls, 5 herds) and after (118 bulls, 19 herds) the natural mating period, and for Campylobacter fetus spp. and Tritrichomonas foetus after the natural mating period (61 bulls, 7 herds). BVDV was detected from an ear-notch sample using a commercially available rapid assay ELISA, bovine herpes virus-1 and T. foetus were screened for by PCR from a penile swab and preputial sample respectively, and C. fetus spp. were screened for by culture of preputial samples. Results None of the bulls tested positive for BVDV antigen. Campylobacter fetus venerealis (or C. fetus fetus) was cultured in 6.6% (4/61) of bulls, representing 2 of the 7 (28.6%) farms that were not vaccinating bulls against bovine genital campylobacteriosis. Bovine herpes virus-1 was identified in 7.8% (10/128) bulls sampled; T. foetus was not identified in any samples. Conclusion Bovine genital campylobacteriosis is present in south-western Victoria, despite longstanding recommendations to vaccinate bulls. Screening bulls for persistent infection with BVDV is probably justified, despite the absence of persistently infected bulls in this study. Further research is warranted to investigate the potential reproductive implications of BHV-1, and the presence of T. foetus. [ABSTRACT FROM AUTHOR]

The ability for protozoan parasites to tolerate pH fluctuations within their niche is critical for the establishment of infection and require the parasite to be capable of adapting to a distinct pH range. We used two host adapted Tritrichomonas foetus isolates, capable of infecting either the digestive tract (pH 5.3–6.6) of feline hosts or the reproductive tract (pH 7.4–7.8) of bovine hosts to address their adaptability to changing pH. Using flow cytometry, we investigated the pH tolerance of the bovine and feline T. foetus isolates over a range of physiologically relevant pH in vitro . Following exposure to mild acid stress (pH 6), the bovine T. foetus isolates showed a significant decrease in cell viability and increased cytoplasmic granularity (p-value < 0.003, p-value < 0.0002) compared to pH 7 and 8 (p-value > 0.7). In contrast, the feline genotype displayed an enhanced capacity to maintain cell morphology and viability (p-value > 0.05). Microscopic assessment revealed that following exposure to a weak acidic stress (pH 6), the bovine T. foetus transformed into rounded parasites with extended cell volumes and displays a decrease in viability. The higher tolerance for acidic extracellular environment of the feline isolate compared to the bovine isolate suggests that pH could be a critical factor in regulating T. foetus infections and host-specificity. [ABSTRACT FROM AUTHOR]